Cervical cancer incidence and mortality. Cervical cancer is the second most commonly diagnosed malignancy and the third leading cause of cancer death in women worldwide. There were 555,100 new cases and 309,800 deaths estimated in 2007, 83% of which occurred in the developing world (American Cancer Society, ACS Global Cancer Facts and Figures, 2007).
In the US in 2010 there were an estimated 12,200 new cases of invasive cervical cancer and an estimated 4,210 deaths from cervical cancer (American Cancer Society, ACS Cancer Facts & Figures, 2010; Jemal A, et al., Cancer statistics, 2010, CA Cancer J Clin 60:277-300, 2010). Since its establishment in 1957, Pap smear has become a routine screening test in the US. As a result, cervical cancer incidence rates have decreased in both white and African American women, pre-invasive lesions of the cervix are detected far more frequently than invasive cancer, and mortality rates have steadily declined as well over the past several decades.
Cervical cancer etiology and pathology. Infection with human papillomavirus (HPV) is a primary etiologic factor in cervical cancer. The magnitude of risk association is greater than that for smoking and lung cancer (Unger E R, Barr E, Human papillomavirus and cervical cancer, in Emerg Infect Dis 10:2031-2032, 2004). Among the 200 HPV types known, HPV16/18 are the most commonly associated to cervical cancer, with more than a 200-fold increased risk (Castellsague X, et al., Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors: implications for screening and prevention, J Natl Cancer Inst 5:303-315, 2006).
However most HPV infections disappear spontaneously and only a small percentage progress to CIN (cervical intraepithelial neoplasia) or CIS (carcinoma in situ). Other risk factors contributing to cervical cancer may be immunosuppression, high parity, smoking, and nutritional factors, as well as long-term use of oral contraceptives (WHO/ICO Information Centre on HPV and Cervical Cancer, Summary report on HPV and cervical cancer statistics in Brazil, 2007; ACS, 2010).
Cervical cancer comprises two major types: squamous cell carcinoma (75%) and adenocarcinoma (20%), affecting the squamous cells and the glandular cells of the cervix epithelium respectively (WHO/ICO, 2007).
The Bethesda system classifies precancerous cervical lesions in: i) atypical squamous cells of undetermined significance (ASCUS); ii) low grade squamous intraepithelial lesions (LSIL) or cervical intraepithelial neoplasia (CIN I), characterized by mild dysplasia; iii) high grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasia, including carcinoma in situ (CIN II, CIN III/CIS) characterized by moderate to severe dysplasia. Note that LSIL/HSIL nomenclature refers to cervical lesions detected by cytology, while CIN nomenclature refers to dysplasia determined upon histological analysis of biopsied cervical tissues (WHO/ICO, 2007).
CIN I rarely (1%) develop into cancer, and mostly return to normal even if untreated; CIN II carry a risk of progression into cancer of 16% by two years and 25% after five years, if left untreated. CIS is cervix confined cancer that will develop into invasive cervical cancer (ICC) over a period of 10 to 12 years. One year and five year relative survival for cervical cancer patients in the US is 88% and 72% respectively, while the 5-year survival rate for patients diagnosed with localized cervical cancer is 92% (ACS, 2010).
Pre-cancerous CIN lesions may be treated by electrocoagulation, cryotherapy, CO2 laser ablation, or local surgery (Campion M, Preinvasive disease. In: Practical Gynecologic Oncology Third Edition. Eds: Berek J S, Hacker N F, Lippincott, Williams & Wilkins, Philadelphia, USA, pp 271-343, 2000; ACS, 2007). Invasive cervical cancers are treated with surgery, radiation or both, as well as chemotherapy in selected cases. Symptoms, often abnormal vaginal bleeding, do not appear until cancer has developed.
Cervical cancer screening. Pap smear screening in developed countries has undoubtedly contributed to the decrease in cervical cancer incidence and mortality, due to early detection of cervical lesions. The Pap test is a cytological staining of cervical cells collected through a simple procedure performed in the doctor's office. Cells are either directly smeared on a slide, then fixed and stained (conventional Pap smear), or first rinsed in a liquid preservative solution to thin mucous and eliminate cell debris, prior to preparing a slide (liquid-based cytology, LBC). In both cases, the slides are read by a cytopathologist.
The Pap smear and LBC methods are based on subjective visual readings of cell morphologies, and have a limited sensitivity of 50% and high susceptibility to intra and inter-individual variability (Boulet G A V, et al., Human papillomavirus in cervical cancer screening: important role as biomarker, Cancer Epidemiol Biomarkers Prey 17:810-817, 2008). 50,000 to 300,000 cells per slide may be read to find 20-30 potentially abnormal cells, and double reading is often required. These tests require highly trained staff and adequate laboratories, making the tests labor-intensive and expensive. So, in addition to limited sensitivity, Pap test is also a relatively costly procedure requiring infrastructure and pathology expertise.
The relatively low sensitivity results in a high false-negative rate, mostly due to inadequate sampling and improper slide preparation. Efforts have been made to address these issues and technologies have been developed that either improve on the way slides are prepared and analyzed, or on the reading burden. The development of LBC technology (ThinPrep, Hologic; SurePath, BD) has provided more standardized method of sampling. For example, the ThinPrep2000 processor purifies cell samples from contaminating blood, bacteria, mucus, and other inflammatory material, prior to depositing it on a slide that will be analyzed by a cytotechnologist. This method detects 65% more LSIL in the general population than conventional Pap smear, and reduces by >50% the number of inadequate cell samples, and provides the ability to perform additional tests out of the same vial. Furthermore, computerized imaging systems now identify suspicious cells that are subsequently examined by a pathologist.
In conclusion, automated slide preparation and automated reading methods have reduced the burden of the analysis, as well as the intra and inter-individual variability of evaluation. Nonetheless, while improving test accuracy, these technologies contribute to increased high-tech infrastructure and cost required for cervical screening, clearly not to the advantage of low infrastructure settings. And still to date the Pap test remains a “reading” based screening, requiring appropriate infrastructure and human resources.
The etiologic relationship between HPV and cervical cancer has been exploited for the development of molecular technologies for viral detection to overcome limitations of cytologic cervical screening. HPV testing by DNA amplification is now used to complement equivocal Pap smears, to triage high risk patients and address them to cytology, and as follow-up after CIN treatment (Boulet, 2008). Currently HPV testing serves as a surrogate end point for cervical cancer screening. However, it has also been suggested for primary screening, particularly in women over age 30, followed by cytology screening if HPV positive (Smith R A, Cokkinides V, Eyre H J. American Cancer Society Guidelines for the Early Detection of Cancer, CA Cancer J Clin 55:31-44, 2005).
According to the ACS recommendations (Smith, 2005), cervical cancer screening should be done every year with Pap test or LBC. If Pap smear is abnormal and reveals ASCUS, HPV testing is done, and if positive, women are referred to colposcopy. If Pap smear reveals LSIL or HSIL, women are immediately referred to colposcopy. Colposcopy is the microscopic examination of the cervix upon acetic acid or Lugol's stain to reveal abnormal cells, which can be in turn biopsied. Women over age 30 who have had three normal Pap test results in a row may get screened every 2-3 years with cervical cytology alone, or every 3 years with an HPV DNA test plus cervical cytology. Women over age 70 and older who have had three or more normal Pap test in a row, and no abnormal Pap test in the last 10 years, may stop screening (Smith, 2005).
Tissue and serum biomarkers. With the aim to improve Pap smear accuracy and complement current cervical screening, potential biomarkers of preneoplastic cervical lesions and cervical cancer have been described.
Antigen Ki-67 is a large nuclear protein, which is expressed in proliferating cells (Goodson W H., et al. The functional relationship between in vivo bromo-deoxyuridine labeling index and Ki-67 proliferation index in human breast cancer, Breast Cancer Res Treat 1998 May; 49 (2): 155-164; Scholzen T., et al. The Ki-67 protein: from the known and the unknown [review], J. Cell Physiol 2000; 182:311-22). Ki-67 is preferentially expressed during all active phases of the cell cycle (late G1-, S-, G2-and M-), but absent in resting cells (G0-). In diagnostic histopathology, antibodies to Ki-67 are used to grade proliferation rates of tumors (Cattoretti G., et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB1 and MIB3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992; 168:357-63). Ki-67 immunostaining using the commercially available antibody MIB-1 has been evaluated as an adjunct test to increase diagnostic accuracy of cervical squamous intraepithelial lesions (LSIL and HSIL; Pirog et al. Diagnostic accuracy of cervical low-grade squamous intraepithelial lesions is improved with MILB-1 immunostaining, Am J Surg Pathol 26:70-75, 2002). Indeed it is reported that there is considerable interobserver variation in the diagnosis of LSIL. MIB-1 immunostaining was found to be more sensitive and specific than HPV testing.
It has been shown that minichromosome maintenance (MCM) proteins, and particularly MCM-2, MCM-5 and MCM-7, are useful for the detection of cervical disease including dysplasia and cancer (Williams et al., Proc Natl Acad Sci U.S.A. 95:14932-14937, 1998; Freeman et al., Clin Cancer Res. 5:2121-2132, 1999), as demonstrated on conventional cervical smears or by immunohistochemical staining of cervical tissues. Recent results using an HPV-transgenic mouse model have shown that MCM-7 further appears to be a specific marker for the detection of high-grade cervical disease by immunochemistry (Brake et al., Cancer Res. 63:8173-8180, 2003; Malinowski et al., Acta Cytol. 43:696, 2004; U.S. Pat. No. 7,632,498 Malinowski et al., 2009).
Cyclin-dependent kinase inhibitor 2A (CDKN2A), also known as p16(INK4a) is a cell cycle regulator overexpressed in cervical preneoplastic lesions harboring HPV16/18 and in cervical cancer. p16(INK4a) overexpression is due to functional inactivation of retinoblastoma Rb protein by HPV E7 protein. p16(INK4a) overexpression is thus related to active HPV gene expression, rather than viral presence only. It has thus been proposed that overexpression of p16(INK4a) may be used as a marker for persistent high-risk HPV infection and detection of high-grade squamous epithelial lesions (HSIL; Klaes et al. Overexpression of p16(INK4a) as a specific marker for dysplastic and neoplastic epithelial cells of the cervix uteri, Int J Cancer 92:276-284, 2001; Agoff et al., p16(INK4a) expression correlates with degree of cervical neoplasia: a comparison with Ki-67 expression and detection of high-risk HPV types, Mod Pathol 16:665-673, 2003; Von Knebel Doeberitz et al, 2004, U.S. Pat. No. 6,709,832).
Immunohistochemical detection of p16(INK4a) in cervical biopsies is in routine use for discriminating between HPV and non-HPV associated lesions (Redman R, et al., The utility of p16(Ink4a) in discriminating between cervical intraepithelial neoplasia 1 and non-neoplastic equivocal lesions of the cervix, Arch Pathol Lab Med 132:795-799, 2008), and as surrogate marker of high-risk HPV infection (Mulvany N J, et al., Diagnostic utility of p16INK4a: a reappraisal of its use in cervical biopsies. Pathology 40:335-344, 2008). Furthermore, p16INK4a immunostaining of ThinPrep cervical specimens is being evaluated for its ability to assist in the identification of high-grade intraepithelial lesions, as assessed by follow-up biopsies and high-risk HPV DNA testing (Meyer et al., Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay, Cancer, 111:83-92, 2007; Doeberitz et al, 2007, U.S. Pat. No. 7,306,926). Finally, an ELISA-based procedure for detecting p16(INK4a) using protein lysates of exfoliative cervical cells has been reported to yield higher sensitivity and specificity for dysplasia and cancer than ThinPrep Test (Ding L, et al., ELISA Test to detect CDK2A (p16(INK4a) expression in exfoliative cells: a new screening tool for cervical cancer, Mol Diagn Ther 12:395-400, 2008).
In conclusion, the use of antibodies against specific biomarkers of cervical cancer and cervical preneoplastic lesions may increase the accuracy of current cervical screening. There is a consensus in the field that misinterpretation and interobserver discrepancies are common, especially in the LSIL cytology category. Indeed antibody immunostaining represents an “objective test”, and insofar may serve as an adjunct to the morphological interpretation offered by the pathologist. Antibodies against specific biomarkers could thus assist in the diagnostic interpretation of LSIL by increasing accuracy of histopathology or cytology-based diagnosis.
Among circulating biomarkers, squamous cell carcinoma antigen serum levels have been found to correlate with tumor stage, tumor size, residual tumor after treatment, recurrent or progressive disease, and survival in squamous cervical carcinoma (Gaarenstroom K N, Bonfrer J M G. NACB, National Academy of Clinical Biochemistry: Guidelines for the use of tumor markers in cervical cancer, 2007). SCC antigen is a group of glycoproteins with molecular weight˜45 KDa, belonging to the family of serine protease inhibitors. There are in fact two genes, SSC1 and SSC2, both located on chromosome 18q21.3, coding for the neutral and acidic isoform respectively. The neutral form is detected in both normal and malignant epithelial cells, whereas the acidic isoform is found in tumor cells and in the sera of cancer patients with well-differentiated squamous cell carcinoma (Kato H, et al., Heterogenous distribution of acidic TA-4 in cervical squamous cell carcinoma; immunohistochemical demonstration with monoclonal antibodies, Jpn J Cancer Res 78:1246-1250, 1987). SSC1 and SSC2 are almost identical, only differing in their reactive loops. There is evidence they regulate proteolytic events in both normal and pathological processes, yet have distinct biological functions (De Bruijn H W A, et al., The clinical value of squamous cell carcinoma antigen in cancer of the uterine cervix, Tumor Biol 19: 505-516, 1998). Elevated levels of SCC have also been found in patients with squamous cell carcinoma of other organs (vulva, vagina, head and neck, lung) as well as in patients with benign diseases of the skin (psoriasis, eczema), lung (sarcoidosis), liver and kidney. However, according to the NACB recommendations (Gaarenstroom, 2007), SCC is not a sufficiently sensitive biomarker for cervical cancer screening, and its clinical utility in prognosis and monitoring response to treatment needs further evaluation.
In conclusion, there is a need to improve the accuracy, performance and cost of current cervical screening. The device and methods of the present invention have utility application as novel cytology-based high throughput cervical screening.